Layer‐by‐layer interleukin‐12 nanoparticles drive a safe and effective response in ovarian tumors

Abstract Ovarian cancer is especially deadly, challenging to treat, and has proven refractory to known immunotherapies. Cytokine therapy is an attractive strategy to drive a proinflammatory immune response in immunologically cold tumors such as many high grade ovarian cancers; however, this strategy has been limited in the past due to severe toxicity. We previously demonstrated the use of a layer‐by‐layer (LbL) nanoparticle (NP) delivery vehicle in subcutaneous flank tumors to reduce the toxicity of interleukin‐12 (IL‐12) therapy upon intratumoral injection. However, ovarian cancer cannot be treated by local injection as it presents as dispersed metastases. Herein, we demonstrate the use of systemically delivered LbL NPs using a cancer cell membrane‐binding outer layer to effectively target and engage the adaptive immune system as a treatment in multiple orthotopic ovarian tumor models, including immunologically cold tumors. IL‐12 therapy from systemically delivered LbL NPs shows reduced severe toxicity and maintained anti‐tumor efficacy compared to carrier‐free IL‐12 or layer‐free liposomal NPs leading to a 30% complete survival rate.


| INTRODUCTION
Immunotherapy has become an increasingly attractive treatment option for cancer therapy since the approval of the checkpoint inhibitor ipilumimab in 2011. 1 Checkpoint inhibitors have elicited durable complete responses in a broad range of cancers, including some malignancies with previously very poor prognoses. 1,2 However, checkpoint blockade benefits only a minority of patients in most diseases. It is becoming clear that immunosuppressive or immune excluded "cold" tumor microenvironments (TME) play a key role in nonresponsive tumors. 3,4 Ovarian cancer is one such malignancy that often presents as a "cold" tumor [5][6][7] and has been particularly unresponsive to checkpoint inhibition. 8 One method to bring immunotherapy to such immune excluded environments is to use complementary therapeutics to drive lymphocyte infiltration and activation into tumors while preventing immune system arrest using checkpoint inhibition. 3 One class of therapeutics with the potential to drive immune infiltration into "cold" tumors are proinflammatory cytokines such as interleukin-12 (IL-12), which has shown a potent ability to drive lymphocyte infiltration [9][10][11] and cure tumors in preclinical models. 12 However, proinflammatory cytokines tend to be highly toxic when given systemically. Indeed, IL-12 showed very high, schedule-dependent toxicity in clinical trials, including two deaths, [13][14][15] motivating the need for any future IL-12 therapies to have pronounced spatio-temporal control over delivery to keep active concentrations in the TME while limiting its systemic exposure. Many newer delivery methods have been attempted to improve IL-12 therapy, such as gene delivery into the tumor, 16,17 microparticle delivery [18][19][20][21] and hydrogel co-formulations, [22][23][24] but these approaches are limited by a need for local injection directly into the tumor. This limits the usefulness of such treatments in widely disseminated diseases that do not have easily injectable tumors, such as ovarian cancer which often presents as a disseminated multifocal tumor burden throughout the peritoneal cavity, requiring intravenous or intraperitoneal delivery of therapeutics. Thus, there remains a need for spatiotemporally controlled, systemically deliverable, nontoxic IL-12.
One promising route for controlled IL-12 delivery from a systemically deliverable carrier is the use of an engineered nanoparticle (NP).
Systemically administered strategies using simple NP formulations 25,26 have also been attempted, but have failed to control delivery selectively to the tumor microenvironment or significantly reduce toxicity.
However, careful engineering of NP structure and surface chemistry has the potential to eliminate these issues by considering the design criteria for optimal cytokine delivery. For IL-12, these criteria include (1) high loading and release of active IL-12, (2) maintenance of NPs on the surface of tumor cells to ensure availability to membrane-bound IL-12 receptors on nearby lymphocytes, (3) high association with cancer cells, and (4) decreased systemic exposure and toxicity.
We previously developed a NP delivery vehicle engineered to meet these design criteria using the layer-by-layer (LbL) technique to adjust the material properties of the particle. [27][28][29][30][31][32][33] We showed that a liposomal NP with IL-12 bound to the liposomal surface and subsequently covered with a bilayer of poly-L-arginine (PLR) and poly-L-glutamic acid (PLE), termed PLE-IL-12-NP, demonstrated >90% loading efficiency of IL-12, extended (>24 h) localization on the surface of cancer cells, high selectivity for binding to cancer cells over other cell types, and significant antitumor efficacy when administered intratumorally in multiple subcutaneous tumor models at reduced toxicity compared to carrier-free IL-12. 33 Indeed, we demonstrated that surface binding of the labile cytokine is key for this formulation to avoid the high temperatures, high pressures and sonication required to generate uniform liposomes as well as to generate a high loading efficiency. By using surface linkages on liposomes, we demonstrated loading efficiencies much higher than passive loading techniques in polymer particles and incorporated a chemical linkage that can be further leveraged to control kinetics of release in the future. The PLE coating on these particles is used for active targeting of the particles and payload to the tumor which shows increased activity over other, passively target particles. 28,33 These particles also critically demonstrate the ability to anchor to the surface of tumor cells due to the PLE surface chemistry 28,33 which is critical for cytokine activity as compared to more traditional anti-cancer payloads as cytokines must F I G U R E 1 PLE-IL-12-NPs are able to selectively bind to tumor cells and remain localized to cell surfaces, releasing their IL-12 cargo to activate T cells and other immune cells over a 24-h period. These characteristics make PLE-IL-12-NPs a strong candidate for safe and efficacious systemic delivery of IL-12 maintain activity on local immune cells and not cancer cells directly.
Moreover, previous work demonstrates the release of active cytokines, a nontrivial finding for labile protein payloads. 33 These cogent particle designs were demonstrated to be critical for the therapeutic both in previous studies 33 and in the current work.
Achieving systemic delivery of IL-12, and doing so in a realistic ovarian cancer model, are key achievements necessary to generate a translational therapy. In this work, we hypothesize that PLE-IL-12-NPs can also enable the delivery of IL-12 to orthotopic ovarian tumors (Figure 1), which requires systemic delivery due to their presentation as widely disseminated metastases throughout the peritoneal cavity. Because our previous work examining nontherapeutic PLE-layered particles showed association with OVCAR8 ovarian tumors upon systemic administration, 28 it was hypothesized that PLE-IL-12-NPs will also concentrate IL-12 in ovarian tumors. The polymer layers act as a hydrated "shield" to minimize off-target IL-12 exposure in the blood stream or peritoneal fluid while anchoring the NPs to the surface of cancer cells and releasing active IL-12 into the tumor microenvironment. In the current study we used the orthotopic HM-1 and KPCA (an immunologically cold tumor) syngeneic models of ovarian cancer to show that PLE-IL-12-NPs given intraperitoneally or intravenously concentrate IL-12 within disseminated tumors, increase the therapeutic window of IL-12, produce long-term antitumor immune responses, and induce a distinct immunological profile post administration conducive to combination therapy with checkpoint inhibitors. As such we demonstrate the ability to bring the promise of immune treatments to these otherwise refractory tumors by controlling the exposure of toxic, immune infiltrating cytokines within the TME.

| Particle formulation and characterization
NP formulations were manufactured similar to previous studies. 28,33,34 Briefly, single chain IL-12 35 was produced via vector cloning and expression in Expi293 cells (ThermoFisher Scintific). Liposome cores were made via lipid film drying (rotovap) followed by rehydration and pressure driven extrusion to 50 nm particle size (Avestin Liposofast-50). Liposomes were comprised of 5% DGS-NTA (Ni), 65% DSPC, 23.9% cholesterol, and 6.1% POPG by mole for therapeutic NPs. Fluorescent NP were made by lowering DSPC to 60% and adding 5% DOPE for addition of fluorophore. NHS ester fluorophores were added to free amines on DOPE for fluorescently labeled liposomes via overnight reaction at room temperature at pH 8.5 with 5 molar excess dye. Excess dye was removed via tangential flow filtration (TFF). Lipid films were made by drying the indicated lipid mixtures in chloroform by rotovap at 20 mbar for 30 min followed by overnight desiccation under vacuum. 50 nm liposomes were made by first rehydrating films with PBS under sonication at 65 C followed by pressure driven extrusion to desired size (50 nm) at 65 C. IL-12 was added to extruded particles by overnight incubation under agitation at 4 C.
Unreacted IL-12 was removed and buffer was exchanged to water via tangential flow filtration through a 100 kDa membrane (Repligen). IL-12 loading was verified by ELISA after digesting particles with 1% triton and 0.1% BSA. Unlayered control particle synthesis ended here. PLE-IL-12-NPs were layered with PLR by mixing with a 0.1 wt eq solution of PLR under sonication, removing unlayered PLR by TFF. PLE was added in at similar manner at 1 wt eq. Polymers were acquired from Alamanda and adsorption conditions were similar to previous report. 33 Throughout NP manufacture sizes, PDIs and zeta potentials were measured via dynamic light scattering (Malvern ZS90).
Nanoparticles were tested for activity in vitro via their ability to stimulate production of IFN-γ from splenocytes prior to in vivo use. NPs were formulated for systemic injection by mixing 9:1 NP solution:50% Dextrose to make injections isotonic with blood. It is important to note that UL-NPs and PLE-IL-12-NPs for studies were made from the same batch of UL-NPs and the layering process results in an approximate increase in diameter of 30 nm and negligible change in final particle charge. Dosing was done on an IL-12 basis.

| Animal studies
All animal experiments were approved by the Massachusetts Institute of Technology Committee on Animal Care (CAC) and were conducted under the oversight of the Division of Comparative Medicine (DCM). Organs were imaged for NP signal (excitation: 745 nm, emission: 800 nm) via an In Vivo Imaging System (IVIS, Perkin Elmer) immediately after harvest. Organs were frozen immediately following imaging and stored at À80 C. Data were analyzed using Living Image software. Background fluorescence measurements were made for each organ based on signal from dextrose only treated mice. Regions of interest (ROIs) were made around treated organs using the contour ROI setting in Living Image. Total radiant efficiencies (TRE) were measured for each treated organ and corrected by the average radiant efficiency from the matching organ in dextrose treated controls. Percent recovered fluorescence for each organ was then calculated as TRE organ ð Þ P mouse TRE . These % recovered fluorescence values were then normalized by organ weight, similar to previously reported studies. 28

| Cytokine levels in organs
Following biodistribution studies, organs were further processed to extract all protein from individual organs using Miltenyi Biotech gentle MACS Octo Dissociator following recommended protocol for protein extraction. Briefly, organs were placed in M tubes with enough buffer to make a 50 g tissue/ml buffer solution. Buffer used for tissue homogenization was RIPA lysis buffer (Thermofisher #89900) with HALT protease inhibitor cocktail (Thermofisher #78430) and 1% active silicon from Y-30 emulsion (Sigma) for anti-foaming purposes.
Samples were spun at 4000 rcf to remove tissue debris and supernatants were analyzed by ELISA for cytokine content.

| In vivo toxicity tests
To test toxicity, B6C3F1 mice (Jackson Labs 100010) were injected either intravenously via the retro-orbital route or intraperitoneally with varying doses as indicated of PLE-IL-12-NPs, dose matched soluble IL-12, dose matched unlayered NPs or PBS for 5 daily doses and monitored daily for weight change. Serum was collected 3 h after the last dose and assayed for IL-12 and IFN-γ levels via ELISA (Peprotech).

| In vivo efficacy tests
1E06 HM-1 mcherry luc2 tumor cells were inoculated in B6C3F1 mice or 1E06 KPCA tumor cells were inoculated in C57BL/6 mice via intraperitoneal injection. Tumors were allowed to establish for 1 week. Subjects were treated with 5 μg intravenously via the retro-orbital route or 5 μg or 10 μg intraperitoneally of IL-12 in PLE-IL-12-NPs, Unlayered NPs, or carrier-free and compared to PBS controls for five daily doses.
Mice were weighed daily to track toxicity. Serum was collected after the last dose to test for systemic cytokine levels. Mice were tracked for tumor burden twice weekly via IVIS. Mice were sacrificed based on ascites accumulation and/or overall body condition.  To test the biodistribution of IL-12 payload, organs from Figures S1b, and S3 were homogenized and assayed for IL-12 content by ELISA ( Figure S4). Note that this recovered IL-12 includes both the delivered IL-12 and endogenously produced IL-12 in response to therapy and thus compounds itself as IL-12 signaling can drive further IL-12 production. 37 There was a trend toward greater amounts of IL-12 in the tumors and less off-target exposure in the liver from PLE-IL-12-NPs as compared to UL-NPs by the 24 h time point ( Figure S4). This suggests, coupled with previously demonstrated release data, 33 that PLE-IL-12-NPs selectively deliver IL-12 to tumors, while UL-NPs lose the attached IL-12 in circulation which can then traffic as carrier-free IL-12. This suggests that PLE-IL-12-NPs can reduce systemic exposure to IL-12 upon systemic delivery. IFN-γ levels were also measured as an indication of IL-12 activity ( Figure S4b) and followed similar trends to NP distribution.

| PLE-IL-12-NPs expand the therapeutic window of IL-12 delivered systemically
Given the enhanced concentration of IL-12 in tumors and a subsequent reduction in severe toxicity mediated by PLE-IL-12-NPs we next tested the NPs anti-tumor efficacy. Mice were inoculated with orthotopic HM-1 tumors that were allowed to form for 7 days prior to 5 daily treatments with 5 μg of IL-12 given carrier-free, from PLE-IL-12-NPs, or from UL-NPs ( Figure 3a) and compared to controls. Subjects were monitored for severe toxicity during and immediately after dosing by weight changes (Figure S5a,b). These data demonstrate that the PLE-IL-12-NP treated animals were healthier than the carrier-free IL-12, though due to the confounding variable of the presence of F I G U R E 2 IL-12 toxicity in healthy mice. (a), Schematic of dosing scheme in healthy animals. Mice were dosed with 5 μg IL-12 in PLE-IL-12-NPs, UL-NPs, or carrier free and compared to 5% dextrose control. (b), Toxicity of various IL-12 delivery methods administered IV (left) or IP (right) as measured by weight loss during and after dosing.**indicate p < 0.01 ***indicate p < .01 as measured by two-way ANOVA with bonferroni post hoc test across all group N = 5 tumors and ascites, toxicity measured by weight loss of free IL-12 was more muted than in healthy mice ( Figure 2). However, tumor burden as measured by fluorescence signal on IVIS ( Figure S5c,d) and survival ( Figure 3b) showed that PLE-IL-12-NP given IP generated more robust anti-tumor responses than UL-NPs or free-IL-12 and led to long-term survival of one out of three mice.
In contrast to IP delivery, IV delivery showed a more muted antitumor response with PLE-IL-12-NPs, while UL-NPs showed an enhanced response. This reduced response of PLE-IL-12-NPs could be because the tumors in these studies were relatively small and not fully vascularized. UL-NPs release IL-12 more readily in the blood stream over extended times than PLE-IL-12-NPs as evidenced by the toxicity data ( Figure 2) which likely allows for easier access of this released IL-12 as a free cytokine to the tumors as compared to much larger layered NPs that cannot extravasate readily into the tumor tissue. However, the UL-NP delivered IV is also a main route of severe toxicity with one out of four tumor bearing mice treated with UL-NPs succumbing to toxic side effects-thus this approach is not a viable treatment.
A further test was carried out at twice the previous dose of IL-12 to test the limits of PLE-IL-12-NP mediated toxicity when given IP. Significant toxicities occurred at this increased dosing levels in both the carrier-free IL-12 and UL-NP treated subjects regardless of tumor status. In healthy animals, all mice treated with carrier-free IL-12 and 50% of the mice treated with UL-NPs needed to be sacrificed during or immediately after dosing due to severe toxicity as measured by severe (>15%) body weight reduction (Figure 3c). In tumor-bearing mice, both carrier-free and UL-NP IL-12 treatments showed significant severe toxicity ( Figure S5e

| PLE-IL-12-NPs enhance immune activity in tumors upon systemic delivery
We next analyzed the immunological response triggered by PLE-IL12-NPs using flow cytometry. HM-1 tumors were established for 14 days after IP implantation before treating with three daily doses of 10 μg IL-12 equivalent from PLE-IL-12-NPs, UL-NPs, carrier-free IL-12 or dextrose control (Figures 5 and 6). Ascites, tumors, and spleens were harvested 24 h after the final treatment.
Samples were profiled for diverse T-cell phenotypes including CD4/8 subtypes, activity markers, effector/memory markers, and exhaustion markers. Myeloid populations were also assessed including macrophages, dendritic cells (DCs), and myeloid-derived suppressor cells (MDSCs) (gating strategy Figures S6 and S7, complete overview by tissue Figure S8).

| DISCUSSION
In this work we demonstrate that the rational engineering of a NP delivery vehicle using the LbL technique makes significant F I G U R E 4 Survival of mice bearing orthotopic ovarian KPCA tumors (1E06 IP). Tumors were allowed to establish for 7 days prior to beginning five daily treatments at 10 μg equivalent of IL-12 (gray arrows). **indicates a significant increase in median survival relative to dextrose controls evaluated using the Log-Rank test (p < 0.01). N = 4 Dex; N = 5 IL-12, NPs F I G U R E 5 PLE-IL-12-NPs show an equivalent immune response to carrier-free IL-12. Another key finding in the reported work is the enhancement of IL-12 therapy from PLE-IL-12-NPs over UL-NPs. We demonstrate throughout that the engineered LbL NP structure is critical to the reduction of severe toxicity as well as the enhancement of efficacy in these experiments, as a simpler unmodified liposomal particle does not show the same results in both toxicity and efficacy. The LbL particles were designed with thorough consideration of the design challenges required for successful cytokine therapy including efficient protein encapsulation, proficient maintenance of cytokine activity from particles, maintenance of cytokine access to surface receptors within the tumor environment, and selective interaction with tumor F I G U R E 6 PLE-IL-12-NPs engage myeloid cells and checkpoint inhibition in the tumor and spleens. HM-1 tumors were allowed to establish for 14 days before dosing with 10 μg IL-12 IP in PLE-IL-12-NPs, UL-NPs, or carrier-free and compared to vehicle control. Tissues were harvested for analysis 24 h after the third daily dose. (a-f) Immune populations found within the tumor environment (a,b,c,e) and spleen (d,f) as measured by flow cytometry. Statistical differences were measured by the Student's t test. *p < 0.05; **p < 0.01; ***p < 0.001. N = 6 (Dextrose, ULNP, LNP), N = 5 (Free IL-12) unless reduced due to insufficient events during analysis cells to concentrate both NP and payload in the tumor to achieve active levels of signaling in the tumor, a critical consideration for IL-12 success, 39 while preventing off target activity which leads to toxicity.
Perhaps most importantly, in this work we demonstrate that PLE-IL-12-NPs are capable of pronounced single-agent efficacy in ovarian tumors driving robust infiltration of anti-tumor immune cells into relatively "cold" tumors. This is a critical finding in ovarian tumors as they have been refractory to most immunotherapy strategies to date. Toxicity concerns in ovarian cancer patients are typically elevated by severe comorbidities, often limiting the application of checkpoint inhibitors, much less proinflammatory cytokines.
Herein we demonstrate that PLE-IL-12-NPs are capable of a nontoxic increase in proinflammatory immune activity in ovarian cancer ( Figure 3) and this increase is maintained even in a demonstrated "cold" tumor environment ( Figure 4). Indeed, we show that this increased proinflammatory immune response within the tumor is capable of a pronounced single-agent response in these previously refractory tumors. However, there is potential for further success in these difficult to treat tumors through combination with checkpoint inhibition or other immunotherapies, which has been deemed a promising path forward for improving immune outcomes. [40][41][42] As IL-12 delivery drives the proinflammatory immune response within the tumor, so too is the exhaustion of T cells driven, as demonstrated herein ( Figure 5). By adding a checkpoint inhibitor such as an antibody against PD-L1 43 or TIM3, both of which showed marked increases after IL-12 therapy, a further improvement in anti-tumor response is conceivable. Beyond combination with checkpoint inhibitors, combination therapy with additional cytokines is also likely to improve this therapy. 42 Finally, in this study we focus on IL-12 delivery from the described LbL-NPs; however, this design is modular and could easily enable the delivery of other synergistic cytokines as well as combinations of cytokines within the same NP construct.

DATA AVAILABILITY STATEMENT
The data for this study are available within the article, with additional data available in the Supporting Information.